Itch and pain are two distinct sensations. Although our previous study suggested that gastrinreleasing peptide receptor (GRPR) is an itch-specific gene in the spinal cord, a long-standing question of whether there are separate neuronal pathways for itch and pain remains unsettled. Here we selectively ablated lamina I neurons expressing GRPR in the spinal cord of mice. These mice showed profound scratching deficits in response to all of the itching (pruritogenic) stimuli tested, irrespective of their histamine-dependence. In contrast, pain behaviors were unaffected. Our data also suggest that GRPR + neurons are different from the spinothalamic tract (STT) neurons which have been the focus of the debate. Together, the present study suggests that GRPR + neurons constitute a long-sought labeled line for itch sensation in the spinal cord.Itch has long been considered to be a sub-modality or sub-quality of pain (1-4), because both sensations share many similarities (5). Whether itch and pain, two distinct sensations, are mediated by distinct neural circuits has been the subject of controversy (6-8). In the spinal cord, arguments for the "labeled line" came from electrophysiological recordings in cat showing the presence of a small subset of histamine-responsive, mechanically, thermally and mustard oil insensitive lamina I STT neurons (9). Recent studies in primates, however, found that histamine-sensitive STT neurons were all responsive to noxious mechanical and chemical stimuli, notably capsaicin, arguing against the "labeled line" for itch (10,11). Although our previous data suggested that GRPR is an itch-specific gene in the spinal cord (12), they could not be extrapolated to imply that GRPR + neurons are itch-specific, simply because neurons expressing one sensory modality-specific gene may also express other sensory modalityspecific genes as often seen in sensory neurons (13). One way to address this issue is to selectively ablate a subset of itch-signaling neurons and assess whether pain behaviors are altered in the absence of these neurons. We selectively ablated GRPR + neurons in the spinal cord of mice by intrathecal administration of bombesin-saporin (bombesin-sap), a toxincoupled to bombesin that binds with high affinity to GRPR and results in GRPR internalization and cell death ( fig.S1) (14,15).We first determined the optimal dose and time course of bombesin-sap treatment. Ablation of GRPR + neurons reduced pruritogen-induced scratching behaviors in a dose-dependent manner ( fig. S2). Most of GRPR + neurons (>75%) were lost two weeks after single intrathecal injection of bombesin-sap (400 ng, Fig. 1. A to C). To determine the specificity of bombesin-sap treatment, we analyzed several subpopulations of neurons in the spinal cord by using laminaspecific molecular markers. Expression of neuromedin U receptor 2 (NMUR2) and prodynorphin was not affected in lamina I of mice treated with bombesin-sap ( Fig. 1. D We next examined scratching behaviors of mice treated with bombesin-sap in response to intradermal i...

Netrins are a family of secreted molecules that are important for axonal outgrowth and guidance in the developing nervous system. However, the signaling mechanisms that lie immediately downstream of netrin receptors remain poorly understood. Here we report that the netrin receptor DCC (deleted in colorectal cancer) interacts with the focal adhesion kinase (FAK), a kinase implicated in regulating cell adhesion and migration. FAK was expressed in developing brains and was localized with DCC in cultured neurons. Netrin-1 induced FAK and DCC tyrosine phosphorylation. Disruption of FAK signaling abolished netrin-1-induced neurite outgrowth and attractive growth cone turning. Taken together, these results indicate a new signaling mechanism for DCC, in which FAK is activated upon netrin-1 stimulation and mediates netrin-1 function; they also identify a critical role for FAK in axon navigation.

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The differentiation and migration of superficial dorsal horn neurons and subsequent ingrowth of cutaneous afferents are crucial events in the formation of somatosensory circuitry in the dorsal spinal cord. We report that the differentiation and migration of the superficial dorsal horn neurons are regulated by the LIM homeobox gene Lmx1b, and its downstream targets Rnx and Drg11, two transcription factors implicated in the development of dorsal horn circuitry. An analysis of Lmx1b mutants shows that Lmx1b normally acts to maintain the expression of the Ebf genes and to repress the Zic genes. Lmx1b mutants also exhibit the disruption of the cutaneous afferent ingrowth, suggesting that the dorsal horn cells might provide important cues guiding sensory axons into the dorsal spinal cord. Our results thus indicate that Lmx1b has a pivotal role in genetic cascades that control the assembly of circuitry in the superficial dorsal horn.

The transient receptor potential channels (TRPs) respond to chemical irritants and temperature. TRPV1 responds to the itch-inducing endogenous signal histamine, and TRPA1 responds to the itch-inducing chemical chloroquine. We showed that, in sensory neurons, TRPV4 is important for both chloroquine-and histamine-induced itch and that TRPV1 has a role in chloroquine-induced itch. Chloroquine-induced scratching was reduced in mice in which TRPV1 was knocked down or pharmacologically inhibited. Both TRPV4 and TRPV1 were present in some sensory neurons. Pharmacological blockade of either TRPV4 or TRPV1 significantly attenuated the Ca2+ response of sensory neurons exposed to histamine or chloroquine. Knockout of Trpv1 impaired Ca2+ responses and reduced scratching behavior evoked by a TRPV4 agonist, whereas knockout of Trpv4 did not alter TRPV1-mediated capsaicin responses. Electrophysiological analysis of human embryonic kidney (HEK) 293 cells coexpressing TRPV4 and TRPV1 revealed that the presence of both channels enhanced the activation kinetics of TRPV4 but not of TRPV1. Biochemical and biophysical studies suggested a close proximity between TRPV4 and TRPV1 in dorsal root ganglion neurons and in cultured cells. Thus, our studies identified TRPV4 as a channel that contributes to both histamine- and chloroquine-induced itch and indicated that the function of TRPV4 in itch signaling involves TRPV1-mediated facilitation. TRP facilitation through the formation of heteromeric complexes could be a prevalent mechanism by which the vast array of somatosensory information is encoded in sensory neurons.

Opioids remain the most effective analgesics despite their potential adverse effects such as tolerance and addiction. Mechanisms underlying these opiate-mediated processes remain the subject of much debate. Here we describe opioid-induced behaviors of Lmx1b conditional knockout mice (Lmx1b f/f/p ), which lack central serotonergic neurons, and we report that opioid analgesia is differentially dependent on the central serotonergic system. Analgesia induced by a opioid receptor agonist administered at the supraspinal level was abolished in Lmx1b f/f/p mice compared with their wild-type littermates. Furthermore, compared with their wild-type littermates Lmx1b f/f/p mice exhibited significantly reduced analgesic effects of and ␦ opioid receptor agonists at both spinal and supraspinal sites. In contrast to the attenuation in opioid analgesia, Lmx1b f/f/p mice developed tolerance to morphine analgesia and displayed normal morphine reward behavior as measured by conditioned place preference. Our results provide genetic evidence supporting the view that the central serotonergic system is a key component of supraspinal pain modulatory circuitry mediating opioid analgesia. Furthermore, our data suggest that the mechanisms of morphine tolerance and morphine reward are independent of the central serotonergic system. serotonin ͉ pain ͉ conditioned place preference ͉ behavior ͉ mouse

There was an error published in Development 131, 3693-3703.The incorrect image (a duplicate of Fig. 5L) was shown in Fig. 5E. A correct image of Zic2 expression in a wild-type E14.5 mouse embryo is shown below and is representative of several experiments. The findings of this experiment remain unchanged.The authors apologise to readers for this mistake.